Biological insect control agent

ABSTRACT

A recombinant baculovirus for use as an insect control agent having a genome which comprises a polyhedrin gene and a heterologous gene, which expresses an insecticidal protein, wherein the polyhedrin gene and the heterologous gene are not under the control of the same promoter, and are located on the genome such that viral progeny produced by a recombination event with wild-type baculovirus which are viable do not retain expression of both the polyhedrin gene and the heterologous gene.

The present invention relates to a baculovirus for use as an insectbiological control agent, and more particularly to a baculoviruscomprising a heterologous gene capable of expressing an insecticidalprotein, use of the baculovirus to minimise production of certain viralprogeny, and a method of controlling insect pests.

By insect biological control agent we mean an agent which when broughtinto association with an insect is capable of infecting the insect andinterfering with the normal biochemical and physiological processes andleading ultimately to the disablement or death of the insect.

Baculoviruses constitute one of the largest and most diverse groups ofinsect-pathogenic viruses, and are commonly used as powerful expressionsystems for heterologous proteins. There is now great interest inbaculoviruses as insect biological control agents. In particular, workis being carried out on improving the time the virus takes to kill itshost insect by combining the pathogenicity of the baculovirus with theinsecticidal action of a toxin, hormone, or enzyme which is active oninsects.

A concern over the use of recombinant baculoviruses capable ofexpressing a heterologous insecticidal gene product to produce enhancedand commercially viable levels of insecticidal activity is that therecombinant baculoviruses might compete with wild-type viruses and hencebecome established within the environment, even perhaps taking over fromthe wild-type virus populations. By wild-type baculovirus, we mean anon-recombinant baculovirus. Emphathis is therefore being put ondeveloping recombinant baculoviruses which do not need to persist in theenvironment to deliver an insecticidal effect.

With the objective of reducing the survival capacity of the recombinantbaculoviruses used as insecticides various, more-or-less, complexsystems have been proposed. Each of these proposals has potentialdrawbacks.

For example, Miller at al (Biotechnology for Crop Protection, 1988, Ed.Hedin et al, pp 405) proposed that effective, safe recombinantbaculoviruses could be produced by a co-occlusion method. In this methodrecombinant baculoviruses, which themselves lack the capacity to expressthe polyhedrin gene necessary for the production of the occlusion bodiesrequired to provide environmental stability, are propagated in mixedinfections with wild-type viruses, which provide the polyhedrin protein.Polyhedra containing both recombinant and wild-type virus particles areproduced. The idea behind this proposal was that the co-occlusionprocess would provide a method for delivering a polyhedrin minus (pol⁻)baculovirus, i.e. lacks the functional polyhedrin gene, to the field inan infectious form. Persistence of the co-occluded pol⁻ baculovirus in avirus population is determined by the probability of co-infection ofindividual larvae and cells with both virus types as the virus is passedfrom insect to insect.

Limited field trials of the survival characteristics of co-occludedvirus populations have been reported by Wood et al (see the generalreview--Annu. Rev. Microbiol. (1991), 45, p69-87, in particular page83). Perhaps rather surprisingly the rate of decline of the polyhedrindeficient genomes from the population of baculovirus was found to beslow.

From a production standpoint, preparation of co-occluded viruspopulations will also be technically demanding, requiring very carefullycontrolled dual infection methods, and essentially wasteful, as asignificant proportion of the product is useless with respect todelivering an improved insecticidal effect.

An alternative approach to the use of genetic manipulation to providebaculovirus populations which are genetically deficient in the abilityto make polyhedra is to replicate baculoviruses which lack thefunctional polyhedra gene in a cell line/host which has been geneticallyengineered to express the missing polyhedrin. In use the resultant virusparticles are active per os, but after propagation the wild-type viruscannot produce polyhedrin or occlusion bodies. The progeny of suchinfections have very low stability and/or poor infective capacity.

Drawbacks with this approach are (i) that polyhedrin levels duringnatural baculovirus infections are very large at the time of occlusionbody production, and reproducing such expression levels in cell lineswill be very difficult, and (ii) that the cell line/host must carry thefunctional polyhedrin gene. There is thus a significant likelihood thatthe recombination events would result in the production of viral genomeswhich have retained the capacity to produce the insecticidal protein andacquired the capacity to make polyhedrin. Once released into theenvironment such baculoviruses will behave like a genetically engineeredpolyhedrin plus (pol⁺) virus, i.e. contains the functional polyhedringene.

Wood et al (International Patent Publication No. 93/22442) suggest thatsupply of polyhedrin and the ability to produce occluded viruses is notalways necessary to produce baculoviruses with a high level of per osactivity. So-called "pre-occluded" viruses generated in the nucleus ofcells infected with polyhedrin deficient viruses are reported to beorally active and are expected to have the limited survival propertiesof non-occluded viruses. However, realisation of this approach will bedependent upon the development of formulation techniques which canprotect the "pre-occluded" virions for a sufficient period that there isa high probability of the target pest insect encountering and ingestingthe virus in the field before it has been inactivated. In naturalocclusion bodies polyhedrin fulfils this function, at least in part.

The genome of the baculovirus is a closed circular superhelical moleculeof double-stranded DNA. Variations of the genome structure have beenobserved within a strain. The difference in DNA sequence may be as aresult of reduplication of portions of the genome, deletions ofsequences, and base substitutions. Reiterated DNA sequences have alsobeen found within genomes, for example it is reported that the genome ofAcMNPV possesses five regions which show DNA homology. There have beenseveral reports of intergenic homology among baculoviruses. For example,the polyhedrin gene and a region encompassing the p10 gene are highlyconserved among Nuclear Polyhedrosis Viruses (NPVs) infectingLepidoptera.

There have also been reported cases of insertion of host cell DNAsequences into the viral genome. In particular, cellular DNA has alsobeen reported to be incorporated into the genome of two closely relatedviruses, AcMNPV and Galleria mellonella NPV (GmMNPV). Without wishing tobe bound by any theory it is believed that the homologous regions may beassociated with such genome mutations. More recently, five regions whichmay be associated with insertion of cellular DNA into the genome ofSeMNPV were identified. For a general review see Arif BM `The Structureof the Viral Genome`, pg 26-27, in `Current Topics in Microbiology andImmunology`--The Molecular Biology of Baculoviruses`, Ed. Doerfler andBohm.

It was therefore realised that as well as competition with wild-typeviruses, a problem with release of recombinant baculoviruses may be thatthey would interact with wild-types in homologous recombination eventsleading to novel baculoviruses. In such homologous recombination eventsa region of DNA occuring between two regions of the genome which arehomologous with regions of a genome of a second virus are transferred,or swapped, between the viruses. Although any risks involved in therelease of a recombinant baculovirus would have been assessed beforerelease, the progeny of any such combinations with wild-types may wellhave altered insect host ranges and/or changed virulence for targetpests.

According to one aspect of the present invention there is provided arecombinant baculovirus for use as an insect biological control agenthaving a genome which comprises a heterologous gene and a polyhedringene, which expresses an insecticidal protein, wherein the heterologousgene and the polyhedrin gene are not under the control of the samepromoter and the heterologous gene and the polyhedrin gene aresufficiently spaced apart so as to minimise production of viable viralprogeny of recombination events with wild-type baculovirus which retainexpression of both the polyhedrin and heterologous gene.

According to another aspect of the present invention there is provided arecombinant baculovirus for use as an insect control agent having agenome which comprises a polyhedrin gene and a heterologous gene, whichexpresses an insecticidal protein, wherein the polyhedrin gene and theheterologous gene are not under the control of the same promoter, andare located on the genome such that viral progeny produced by arecombination event with wild-type baculovirus which are viable do notretain expression of both the polyhedrin gene and the heterologous gene.

By viable we mean that the progeny does not lack an essential gene.

The present invention can best be described with reference to thefollowing accompanying drawings:

FIG. 1 shows a schematic representation of a recombination eventinvolving a recombinant virus according to the prior art;

FIGS. 2, 3 and 4 show schematic representations of recombination eventsinvolving a recombinant virus according to the present invention;

FIG. 5a shows a AcUW1 baculovirus which lacks the p10 gene;

FIG. 5b shows a complete AcUW1 baculovirus; i.e., a baculovirus havingthe p10 gene;

FIG. 5c shows a AcUW1 baculovirus having incomplete polyhedra and havinglost virons;

FIG. 5d shows a complete AcUW1 baculovirus; i.e., a baculovirus havingcomplete polyhedra and virons;

FIGS. 6a and 6b show the comparative structural features of the genomesof recombinant baculoviruses AcMNPV/pAcUW21/sKK-0 #1 and AcUW1-PH/sKK-0#2;

FIG. 7 shows the sequence of a synthetic KK-0 (sKK-0) conotoxin geneshowing the regions encoding pre-pro conotoxin KK-0;

FIG. 8 shows the sequence of a synthetic KK-0 conotoxin gene showing thelocation of restriction sites introduced to facilitate cloning and othermanipulations;

FIG. 9 shows the sequence and relationships of oligonucleotides used forthe assembly of a synthetic KK-0 conotoxin gene sKK-0;

FIG. 10 shows the cloning and sequencing strategy for the generation andcharacterisation of pVL1392/sKK-0 recombinant vectors;

FIG. 11 shows the sequence determined for the sKK-0 insert regions oftransfer vector pVL1392/sKK-0 #2.2; and

FIG. 12 shows comparisons of the predicted amino acid sequences of theprecursors of Conus textile KK-0, KK-1 and KK-2 peptides and thepredicted sequences of the corresponding mature peptides.

FIG. 1 shows a schematic representation of a recombination eventinvolving a recombinant virus according to the prior art, wherein F isthe prior art recombinant virus, B is the wild-type virus with which itis recombining, the crosses indicate regions of homology and hencedefine the region of co-transfer, wh⁺ indicates the heterologous geneand pol⁺ indicates the polyhedron gene.

In F, the wh⁺ and pol⁺ genes are in their conventional positions, i.e.the genes are extremely close together. There is very little sequencebetween the genes and therefore very little chance that it will behomologous to another virus, and hence able to act as a substrate forrecombination. It will be appreciated that there is a very much greaterchance of homologous sequences occuring either side of both genes, andthus a correspondingly much greater chance that both genes will movetogether as a unit during recombination.

FIGS. 2 and 3 each show a schematic representation of a recombinationevent involving a recombinant virus according to one aspect of thepresent invention, represented by A, and a wild-type virus, C and Drespectively.

It will be appreciated that the likelihood of co-transfer of both thewh⁺ and pol⁺ genes to another virus by homologous recombination is verymuch less with the recombinant of the present invention than with theprior art recombinant. The separation of the wh⁺ and pol⁺ genes inaccordance with the present invention means that there is much moresubstrate between the genes upon which recombination events could occur,and therefore a correspondingly greater chance that both wh⁺ and pol⁺genes will not be transferred together.

If recombination occurs around the position occupied by the wh⁺ gene inA, as illustrated by FIG. 2, then the resulting A will have two pol⁺genes, but no warhead and the resulting C will have wh⁺, but its lack ofthe pol⁺ gene means that it will be crippled.

If recombination occurs around the position occupied by the pol⁺ gene inA, as illustrated by FIG. 3, the resulting A will have wh⁺, but will becrippled due to the lack of the pol⁺ gene, and the resulting progenyvirus D will not have the warhead.

A further feature of the present invention is that even if wh⁺ pol⁺ areco-transferred, there is a greater likelihood than with the prior artthat the virus to which they are transferred will lose at least oneessential gene in the process, and thus be at least disabled or evennon-viable. This situation is particularly true for co-transfer betweendistantly related viruses. This situation is shown schematically in FIG.4, in which X represents the position of the essential gene. X islocated in different positions on the genone in A and E, such thatrecombination as shown will result in loss of X from wild-type virus E.

Intrinsically a virus according to the present invention is:

safer than a construct based on more wild-type constructs;

safer with respect to likely recombination events; and

straightforward to produce.

Preferably at least 10% of the genome of the baculovirus should separatethe heterologous gene and polyhedrin gene. Even more preferably about12% of the genome separates the two genes. This is illustrated for easeof reference only by FIGS. 6. In FIGS. 6 the location of sequences isreported with reference to the scale of 0-100 map units. AcMNPV has agenome of approximately 130/131 kilo base pairs in size. Therefore,according to the present invention the heterologous gene and thepolyhedrin gene are preferably at least 13 kb , even more preferablyabout 15 kb to about 16 kb , apart.

Generally, the genomes of baculovirus range from about 90 to 200 kb. Itwill therefore be appreciated that the appropriate separation distancebetween the genes will depend on the baculovirus employed in the presentinvention.

Preferably, the polyhedrin gene is under the control of the p10promoter, and the heterologous gene is under the control of any otherpromoter, for example the polyhedrin promoter.

Although this is a preferred embodiment, for the avoidance of doubt wewould mention that the polyhedrin gene could be at any other position inthe genome apart from its natural site, and where it did not disrupt anessential gene. The same is true of the heterologous gene.

It is also proposed that a recombinant which has a polyhedrin gene atthe location normally occupied by the p10 gene, which p10 gene isdisabled or deleted, and can be genetically manipulated to accommodategenes encoding insecticidal proteins at the position which would usuallyhave been occupied by the polyhedrin gene provides a simple solution tothe generation of diminished stability viruses. Such p10 deficientviruses have many features which render them both effective insecticideswhilst still being genetically disabled with respect to wild-typeviruses.

Thus, in accordance with another preferred embodiment of the presentinvention the genome has been modified to disable or delete the p10gene.

Baculoviruses according to the present invention which also lack the p10gene are also found to have the following features:

insects which are infected with such a virus produce low yields of viralprogeny at the time of death compared with insects infected at the samedevelopmental stage with wild-type virus;

the polyhedra produced are small and misshaped when compared to thewild-type polyhedra. They are thus likely to have reduced survivalcapacity;

the polyhedra are however as infectious as wild-type polyhedra meaningthat production will still be straightforward.

This feature of the invention is illustrated in FIGS. 5 (where Mag^(n)=magnification). FIG. 5a shows a AcUW1 baculovirus which lacks the p10gene. By comparison with the p10⁺ AcUW1 baculovirus of FIG. 5b, it willreadily be seen that the polyhedron of the p10⁻ baculovirus isincomplete. A cluster of the disabled p10⁻ AcUW1 baculoviruses is shownin FIG. 5c. This FIG. 5c illustrates that not only are the polyhedraincomplete, but that these baculoviruses have lost virons. Again thedisabled baculoviruses in FIG. 5c can be compared with the healthiernon-disabled baculoviruses shown in FIG. 5d.

In order to prepare these photographs polyhedra were fixed in 30%gluteraldehyde in 0.05M phosphate buffer for 90 minutes at 4° C., washed3 times in buffer, and postfixed in 1% osmium tetroxide in 0.05Mphosphate buffer. Following dehydration and infiltration the sampleswere embedded in Spurr's resin (Spurr, 1969), sectioned and stained in2% alcoholic uranyl acetate for 15 minutes followed by 0.2% lead citratein 0.1N sodium hydroxide for 5 minutes.

A preferred embodiment of the present invention is illustrated ingreater detail by FIG. 6b, whose construction is described in theExamples below. This Figure can be instructively compared with thefeatures of the recombinant baculovirus illustrated in FIG. 6a. FIG. 6aillustrates the conventional location of the heterologous gene, namelyin close proximity to the polyhedrin gene.

Various preferred features and embodiments of the present invention willnow be described by in greater detail.

The present invention will be described for ease of reference inrelation to the use of a conotoxin as the heterologous gene, inparticular, the mollusc and crustacean active "King Kong" conotoxin(Hillyard et al. (1989) Biochemistry), later designated KK-0 SEQ ID 16!(Woodward et al. (1990) EMBO J. 9 1015-1020), and two related peptidesKK-1 SEQ ID 17! and KK-2 SEQ ID 18! reported at the Conferences JacquesMonod on Toxines Animales (Aussois, France) on 24-28 Oct. 1988. Asdetailed in Example 1 below, synthetic peptides corresponding to thereported sequences of KK-0, KK-1 and KK-2 (see FIG. 12) were preparedand assessed by injection into specimens of adult Periplanata americana(Order: Dictyoptera) and larval Heliothis virescens and Trichoplusia ni(Order: Lepidoptera). Both the preparations KK-0 and KK-1 showeddistinct insecticidal and/or paralysis inducing activity in each of thespecies tested. It will also be seen that use was made of a syntheticgene SEQ ID 1! designed to encode an mRNA molecule which would in turnencode the presumed natural precursor of the "King Kong" (KK-0)conotoxin: a 78 amino acid protein which is likely to enter thesecretion pathway during synthesis and may initially be produced as apro-toxin which is subsequently processed to produce mature KK-0, atleast in Conus textile (Woodward et al. (1990) EMBO J. 9 1015-1020). Thesynthetic gene we used for this work was designed without regard for thenatural nucleotide sequence of the Conus textile KK-0 gene. Rather thegene was designed to ensure that, although it encoded the natural KK-0precursor, it was likely to be both efficiently expressed in insect cellsystems and was convenient to manipulate (see details in Example 2).Though we have not therefore used a natural KK-0 gene or cDNA from Conustextile there are, however, nG grounds to anticipate that such asequence, or another synthetic gene capable of encoding the KK-0precursor, would not be equally effective in enhancing the speed ofaction of a baculovirus host. The synthetic gene (sKK-0) we haveemployed actually has only 77% nucleotide sequence identity with thecoding sequence of natural KK-0 mRNA. Further details of this toxin aregiven in our co-pending International Patent Publication No WO 94/23047.

It will be readily appreciated by a person skilled in the art that thepresent invention is applicable to other heterologous genes expressinginsecticidal proteins, which are available or which are developed.Examples of such genes are genes expressing the toxin from the NorthAfrican scorpion Androctonus australis Hector (AaIT), and genesexpressing the Tox 34 toxin from the straw itch mite Pyemotes tritici(Tomalski MD and Miller LK, Nature, (1991), 352, 82-85).

The baculovirus may be Autographa californica Multiply Enveloped NuclearPolyhedrosis Virus (AcMNPV) or any other available or developed viralspecies which is suitable for genetic engineering or manipulationtechniques. Further examples include Bombyx mori NPV, Spodoptera exiguaMNPV, Galleria mellonella MNPV, Trichoplusia ni MNPV, Choristoneurafumiferana MNPV, Orgyia pseudotsugata MNPV, Spodootera frugiperda MNPV,Mamestra brassicae MNPV, the so-called Celery Looper virus, designatedHPV-85-CLMEV, mentioned in International Patent Publication No.W090/10387, the so-called NC-1 virus mentioned in our co-pendingInternational Patent Application No. PCT/GB94/02438 and Heliothis zeaSNPV (Single Enveloped Nuclear Polyhedrosis Virus).

Any pol⁻ transmission vector suitable for use in connection with themodification of the baculovirus genome may be used in preparing theinsect control agent of the present invention. Examples include pVL1392and pVL1393. Other suitable vectors are known in the art.

A plasmid based on pVL1392 incorporating the DNA sequence of FIG. 7 andFIG. 8 has been introduced into Escherichia coli and deposited in theNational Collection of Industrial and Marine Bacteria, Aberdeen UK,under the number NCIMB 40540, on 12 Mar. 1993.

A recombinant baculovirus according to the present invention can beconstructed in accordance with known techniques. In general terms anappropriate transfer vector is constructed, as mentioned above. Thistransfer vector contains appropriate homologous sequences to the DNAsequences in the wild-type viral genome. Upon cotransfection of viralDNA and the plasmid vector, recombinant progeny are likely to arise dueto homologous recombination. The recombinant viruses can then beseparated from the parental viruses using conventional techniques.

Various preferred aspects of the invention will now be illustrated bymeans of the following Examples.

EXAMPLE 1

Biological Assessment of Synthetic Preparations of "King Kong" (KK-0),KK-1 and KK-2 Peptides

Preliminary assessments of the biological activity of syntheticpreparations of KK-0, KK-1 and KK-2 peptides were made in a series ofinjection studies using adult cockroaches (Periplaneta americana:Blattidae: Dictyoptera) and fifth-instar tobacco budworm larvae(Heliothis virescens: Noctuidae: Lepidoptera).

The proteins proved to be insoluble in distilled water and the standardaqueous buffers normally used for injection. KK-0 and KK-1 weresuspended at a concentration of 10mg/ml in 0.1M ammonium bicarbonate.After gentle agitation for 1 hour the suspensions were filtered througha 0.2 μm filter. A visual assessment indicated that approximately 50% ofthe KK-0 product was solubilised whereas substantially less of the KK-1product dissolved. This made it difficult to accurately quantify andcompare dose rates directly for the different proteins. However, it waspossible to estimate the maximum doses delivered. KK-2 was extremelyhydrophobic and was dissolved in DMSO at a concentration of 10 mg ofcrude product per ml.

Tobacco budworm larvae (TBW) were injected using a 10 μl Hamiltonsyringe fitted with a 15 mm 33 gauge needle. Early fifth-instar larvaewith an average body mass of approximately 300 mg were injected with 1-4μl of each treatment. Typically, there were at least 5 replicates ofeach treatment. Adult male cockroaches were injected using a 50 μlHamilton syringe fitted with a 15 mm 27 gauge needle. Each animalreceived a dose of 1-10 μl of test solution; average body mass wasapproximately 840 mg. Control insects were injected with an equivalentvolume of the appropriate solvent.

After injection, treated insects were held individually in containerswith a food supply under controlled conditions (25° C., 65% RH) for upto 72 hours. Observations were made periodically to check for anyunusual symptomology.

Tobacco budworm

Injection of both KK-0 and KK-1 into H.virescens larvae resulted in acharacteristic "flaccid paralysis" (Table I). Typically, symptoms wereobserved almost immediately after injection with larvae appearing tobecome "narcotised". Affected larvae were unable to stand and/or take acoordinated step. Furthermore, they were unable to reinvert when placedupon their back and were capable only of limited feeble movements of themouthparts and claspers in response to stimulation (gentle prodding witha wooden cocktail stick). At the peak of the "narcosis", affected larvaewere limp and pliable. These effects were relatively short-lived,however, and full recovery had normally occurred by 5-10 minutes afterinjection. All larvae were alive and well at 24 hours after treatment(24 HAT). Injection of higher doses led to an increase in both theseverity and duration of the symptoms. The symptomology appeared to bemore pronounced with KK-0.

No abnormal effects were observed following injection of KK-2 proteininto H.virescens larvae at a single rate equivalent to a maximum dose ofapproximately 10 μg protein per larva (ie 33 μg protein/mg larva).

Cockroaches

All three proteins produced distinctive and debilitating effects incockroaches (Table II). In each case, the severity of the effectsincreased with the volume of protein suspension injected.

Injection of KK-0 protein suspension into cockroaches initially causedsevere tremoring followed by a loss of coordination, paralysis andknockdown. The effects were observed initially in the back legs butrapidly spread to the middle and finally the front legs leading to thecollapse of the insect. Other symptoms included dorsal arching. Affectedinsects failed to recover and were dead by 22 HAT. Insects injected withthe KK-1 protein showed similar symptomology although the effectsappeared to be slightly less severe at the lowest dose of 1 μl ofprotein suspension.

Abnormal effects were also observed briefly in insects injected with 2and 5 μl of the KK-2 peptide. Symptoms included arching of the back,flattening of the wings and loss of coordinated movement and occurredwithin a few minutes of injection. The effects appeared to be transitoryand affected insects appeared to have fully recovered by 20 minutespost-injection. However, all animals in these treatments were dead at 72HAT. No unusual symptoms were recorded in insects treated with thelowest rate (1 μl volume) and all insects were alive and well at 72 HAT.

These observations were confirmed in subsequent experiments anddemonstrated that the conotoxin proteins have insecticidal activityagainst cockroaches and TBW larvae.

EXAMPLE 2

Synthetic King Kong (sKK-0) Conotoxin Gene Design

Since there was no a priori reason to believe that the codon usage ofthe natural KK-0 gene would be particularly advantageous for expressionin insect cells, we decided to design a novel synthetic (sKK-0) gene.This was undertaken with the aid of such PCGene™ (Intelligenetics, 700East Camino Real, Mountain View, Calif.) programmes as TRANSL, RESTRIand MUTSITE.

Features of the sKK-0 gene were that:

It encoded precisely the amino acid sequence SEQ ID 2! for the KK-0propeptide described by Woodward et al. through their cDNA cloningstudies ((1990) EMBO J. 9 1015-1020).

The codons used to encode the KK-0 propeptide were selected because theyare favoured or, at least, frequently encountered in insects (Drosophilamelanogaster) and yeast (Saccharomyces cerevisiae) since it wasanticipated that expression studies might also be undertaken in thelatter organism (Ashburner et al. (1984) Develop. Genetics 4 295-312;Ikemura (1985) Mol.Biol.Evol. 2 13-34; Sharp (1986) Nucleic AcidsResearch 14 5125-5143).

The nucleotide sequence immediately preceding the translation initiationcodon (ATG) matches closely the preferred sequence for animal mRNAsdefined by Kozak ((Kozak (1981) Nucleic Acids Research 9 5233-5252;Kozak (1984) Nucleic Acids Research 12 857-873; Kozak (1986) Cell 44283-292; Kozak (1987) J.Mol.Biol. 196 947-950).

No account at all was taken of the codons used by Conus textile toencode the KK-0 propeptide. (As a result the overall homology of sKK-0and natural KK-0 genes is only 77.2%).

Flanking restriction enzyme recognition sites (upstream BglII anddownstream XmaIII and BamHI) were incorporated into the design tofacilitate direct cloning into the initially chosen baculovirus transfervector pVL1392 (InVitrogen Corporation, 3985 Sorrento Valley Boulevard,San Diego, Calif.). Cloning with these enzymes was expected to result inthe potential to transcribe the gene directly from the AcMNPV polyhedrinpromoter carried by this transfer vector (see FIG. 10).

Several additional unique hexanucleotide recognition sequence sites wereincorporated into the sKK-0 sequence at positions throughout the genebut without affecting the amino acid sequence encoded. These sites wereincluded to facilitate recombinant characterisation and possiblesubsequent manipulations of the gene (see Example 14). None of the sitesincorporated would result in the inclusion of particularly unfavourablecodons.

The resultant sKK-0 sequence SEQ ID 1! is shown in FIG. 7, highlightingthe coding sequence for the KK-0 propeptide, and in FIG. 8, highlightingthe position of restriction enzyme recognition sites.

EXAMPLE 3

Synthesis of an sKK-0 Gene

The strategy selected for assembly of the sKK-0 gene involved synthesisof overlapping complementary oligonucleotides encoding the DNA fragmentshown in FIGS. 7 and 8 followed by DNA polymerase mediated completion ofthe single stranded regions and polymerase chain reaction (PCR)amplification to generate abundant quantities of the complete genefragment (see FIG. 9).

Thus 4 oligonucleotides (ConoA, ConoB, ConoC, ConoD) SEQ ID 3, 4, 5, 6!,which together encode the complete sKK-0 DNA fragment, were synthesisedby standard methods on an Applied Biosystems 380B DNA Synthesiser. Twoadditional shorter oligonucleotides, ConoPCR1 & ConoPCR2 SEQ ID 7, 8!,which are identical to the 5' 30 and 28 nucleotides of ConoA and ConoDwere also synthesised to use as PCR primers. Each oligonucleotide wasdeprotected by incubation for approx. 8 hours at 55° followed by dryingdown under vacuum. They were then dissolved in 200 μl TE buffer (10 mMTris/HCl (pH 8.0), 1 mM EDTA), concentrations measured by UVspectroscopy (1 OD₂₆₀ unit/ml. equivalent to approximately 20 μg/ml) anddiluted with TE to give approximately 10 μM stocks. These were stored at-20° C. until use. sKK-0 synthesis reactions then comprised:

1) Mixing 5 μl each of 10 μM ConoA and ConoB in a sterile 500 μl conicalpolypropylene microfuge tube together with 5 μl 10 mM dGTP, 10 mM dATP,10 mM dCTP, 10 mM TTP and 5 μl 500 mM KCl, 10 mM Tris-HCl (pH 8.5), 15mM MgCl₂ 0.1% gelatine, making up to 49 μl with water and adding 1 μl (5units) Taq DNA polymerase (Perkin/Elmer Cetus) before overlaying with 2drops of light paraffin oil (Sigma). The tube was then capped, placed ina Techne PHC-1 Programmable Dri-Block® and subject to the followingtemperature cycle:

    ______________________________________            1.1'        @ 94° C.            1'          @ 55° C.            4'          @ 73° C.    ______________________________________

5 times, followed by a final incubation period of 7'@73° C.

2) Mixing 5 μl each of 10 μM ConoC and ConoD and treating exactly as in(1) above.

3) Taking 0.5 μl reaction (1) products mixing with 0.5 μl reaction (2)products and 5 μl reaction (1) products mixed with reaction (2)products. To each mixture was then added 10 μl 10 μM ConoPCR1; 10 μl 10μM ConoPCR2; 10 μl 500 mM KCl, 100 mM Tris-HCl (pH 8.5), 15 mM MgCl₂,0.1% gelatine; 10 μl 10 mM dGTP, 10 mM dATP, 10 mM dCTP, 10 mM TTP, andthen making up to 99 μl with water before adding 1 μl (5 units) Taq DNApolymerase (Perkin/Elmer Cetus) and finally overlaying with two drops oflight paraffin oil. In parallel, control reactions were set up whichlacked PCR primers, reaction 1 or reaction 2 products. The reactionswere then subject to the following temperature cycle in the TechneProgrammable Dri-Block®:

    ______________________________________            1.1'        @ 94° C.            1'          @ 68° C.            4'          @ 73° C.    ______________________________________

25 times, followed by a final incubation period of 7'@73° C.

Analytical agarose gel electrophoresis on 10 μl aliquots of reaction 3products then confirmed that only in those reactions which had containedall necessary DNA fragments and oligonucleotides had an approx. 270 bpfragment been produced. The remainder of each DNA fragment was thenworked up by phenol extraction with an equal volume of water saturatedphenol (twice) and then an equal volume of water saturated n-butanol(twice) before recovery by ethanol precipitation.

EXAMPLE 4

Assembly of pVL1392/sKK-0 Recombinant Transfer Vectors

After recovery, each of the above PCR products was subject to digestionwith BglII and EclXI (an isoschizomer of XmaIII) restriction enzymesaccording to the manufacturers recommendations. In parallel an approx. 5μg aliquot of pVL1392, a commercially available baculovirus (AcMNPV)transfer vector (InVitrogen), was similarly treated. Restriction digestswere then run on a preparative 0.8% agarose/Tris-acetate electrophoresisgel containing 0.5 μg/ml ethidium bromide. The PCR products and thelinearised vector DNA fragments were excised from the gel under UVillumination. They were then recovered by centrifugation throughsiliconised glass wool and ethanol precipitation. Aliquots of theisolated insert and vector DNA fragments were then mixed, together withT4 ligase in the appropriate buffer conditions (Sambrook J., Fritsch E.F. & Maniatis T. (1989) "Molecular Cloning: A Laboratory Manual". ColdSpring Harbor Press.). The ligation mixtures were then used to transformcompetent DH5α cells by standard methods (Sambrook et al. ibid.).Progeny transformants were selected by overnight growth on L agar platescontaining 100 μg/ml ampicillin.

Transformants were then screened for the presence of sequencescomplementary to the sKK-0 gene by colony hybridisation on HyBond-N(Amersham International) filters by standard procedures (Sambrook et al.ibid.). The hybridisation probe used was oligonucleotide ConoB, whichhad been 5' phosphorylated by treatment with g³² P-ATP (AmershamInternational) and T4 polynucleotide kinase (Northumbria BiologicalsLtd.). Plasmid DNA was prepared from six strongly hybridising colonies.Restriction analysis of these DNAs suggested that three were pVL1392derivatives containing inserts with approximately the size and featuresexpected for sKK-0.

The sequence of the inserts present in the selected pVL1392 recombinants(#2.2, #5.2 & #6.1) was then checked using a Sequenase™ (USB, Cleveland,Ohio) kit and two synthetic oligonucleotide primers (pVL1392FOR andpVL1392REV) SEQ ID 13, 14! designed to hybridise to the regions ofpVL1392 flanking the BglII and XmaIII recognition sites and to allowsequencing of any insert introduced between those sites (see FIG. 10).The sequence of the insert SEQ ID 15! in one of the three recombinants(#2.2) matched precisely that expected for sKK-0 cloned in the expectedmanner (see FIG. 11). The other plasmids contained inserts with minorsequence variations (mutations).

Recombinant #2.2, a derivative of transfer vector pVL1392 containinggene sKK-0 under the transcriptional control of the AcMNPV polyhedrinpromoter, was therefore selected for all subsequent studies. An E.coliDH5α culture carrying recombinant plasmid pVL1392/sKK-0 #2.2 has beendeposited in the National Collection of Industrial and Marine Bacteria,Aberdeen, UK as deposit number NCIMB 40540.

EXAMPLE 5

Generation of Recombinant (Polyhedrin Minus) AcMNPV Carrying sKK-0

Using standard baculovirus techniques, as described for example in King& Possee (1992) "The Baculovirus Expression System: A Laboratory Guide"(Chapman & Hall), recombinant AcMNPV virus carrying sKK-0 under thetranscriptional control of the polyhedrin promoter, but lacking afunctional polyhedrin gene, were generated by co-transfection of Sf21cells with approx. 200 ng. SauI (Boehringer, Mannheim) linearisedAcMNPV.lacZ (Possee & Howard (1987) Nucleic Acids Research 1510233-10248) and 1 μg pVL1392/sKK-0 #2.2 DNA. Selection of recombinantvirus was initially based on the inability of recombinant lacZ- virus tometabolise X-gal (GIBCO/BRL, Grand Island, N.Y.) in plaques generated inSf21 cell monolayer culture. Six such lacZ- virus ("CONO-A", "CONO-B","CONO-C", "CONO-D", "CONO-D" and "CONO-E") were picked and replated onSf21 cells to purify them to homogeneity and confirm their inability tosynthesise βgalactosidase. A small scale (approx. 4 ml) culture of eachpurified virus was then prepared by seeding 1.5×10⁶ Sf21 cells in 4 ml.TC100/7.5% foetal calf serum medium (both from GIBCO/BRL) in a 25 cm²tissue culture flask (Bibby, Stone, Staffs.)), incubation overnight at28° C., addition of 50% of the virus recovered by picking a singlepurified plaque and continuing incubation at 28° C. for a further 6days. Aliquots of each of these virus stocks were then taken forbioassay (see Example 6). Parallel aliquots were used to prepare smallsamples of viral DNA for physical analysis (King & Possee (1992) ibid.).

Diagnostic physical analyses undertaken with the prospectivesKK-0/AcMNPV recombinants were:

a) PCR studies with sKK-0 and, in parallel, βgalactosidase gene specificprimers.

b) Southern blot analysis on viral DNA treated with ClaI and BglII,fractionated by electrophoresis on a 1% agarose gel, transferred toHyBond-N filters and hybridised with an isolated, random-primed α³²P-dCTP labelled, sKK-0 probe.

These studies confirmed that virus "CONO-C" contained an sKK-0 gene,with all the expected physical features, and lacked a βgalactosidasegene. The other prospective recombinant viruses lacked the sKK-0 gene.

EXAMPLE 6

Bioassay of Prospective AcMNPV/pVL1392/sKK-0 Recombinant Virus

The biological activity of putative AcMNPV/pVL1392/sKK-0 viruses wasevaluated in a series of injection studies using late-stage Heliothisvirescens larvae. Aliquots of purified non-occluded virus were injectedinto fourth- or fifth-instar H.virescens larvae using a 10 μl Hamiltonsyringe fitted with a 15 mm 33 gauge needle. Typically, at least 5larvae were injected per treatment with a standard volume of 1 μl volumeof virus suspension. Control insects were injected with an equivalentvolume of AcMNPV/pVL1392/lacZ virus, ACMNPV wild type virus or sterilewater. After injection, larvae were held individually on artificial dietand examined at 24-hour intervals thereafter for a further 7 days. Oneach assessment occasion the number of live and dead larvae wererecorded. Surviving larvae were examined for any unusual symptomologyand/or behavioural responses.

Initially, aliquots of the six putative AcMNPV/pVL1392/sKK-0 clonesgenerated as described in Example 5 were tested. Only one,AcMNPV/pVL1392/sKK-0 CONO-C, exhibited any unusual effects. Thebiological observations confirmed the physical analyses described inExample 5 which indicated that only the CONO-C virus carried the sKK-0gene with all the expected physical features. Further injection assaysto characterise the biological properties of AcMNPV/pVL1392/sKK-0 CONO Cwere carried out using titred stocks of the virus.

Data comparing the biological efficacy of AcMNPV/pVL1392/sKK-0 CONO-Cand AcMNPV/pVL1392/lacZ viruses against early fourth-instar H.virescenslarvae are presented in Table III. At 72 hours after treatment (72 HAT),4/11 larvae treated with the AcMNPV/pVL1392/sKK-0 CONO C showed eitherfull or partial flaccid paralysis. Larvae classified as "fullyparalysed" were moribund: unable to stand, reinvert or walk and capableonly of very feeble movements of the mouthparts and claspers in responseto stimulation. "Partially paralysed" larvae showed local paralysis ofthe mid and posterior regions of the body although the anterior regionincluding the head and mouthparts could respond normally to stimulation.These larvae could reinvert when placed on their backs and were capableof limited locomotion although movements were slow and poorlycoordinated. At 96 HAT, all larvae treated with the AcMNPV/pVL1392/sKK-0CONO C virus were either dead or showing abnormal symptomology. Incontrast, all larvae injected with the AcMNPV/pVL1392/lacZ control viruswere alive and behaving normally at 96 HAT; 1/10 of these larvae haddied at 120 HAT and 6/10 larvae were dead at 144 HAT. AcMNPV/pVL1392derivatives carrying the lacZ gene have a slower speed of kill than thewild type virus. Typically, the wild type AcMNPV starts to causesignificant mortality at 120 HAT and beyond. Thus it may be concludedthat the AcMNPV/pVL1392/sKK-0 CONO C construct has a faster speed ofaction and hence an enhanced insecticidal effect than both the AcMNPVwild type and AcMNPV/pVL1392/lacZ control viruses.

EXAMPLE 7

Assembly of pAcUW21/sKK-0 Recombinant Transfer Vectors

With the objective of preparing polyhedrin⁺ (occluded) recombinantAcMNPV/sKK-0 derivatives, which would be infectious per os and hencecapable of realistic assessment for dose response effects and forevaluation for crop protective effects, we chose initially to employ thepAcUW21 transfer vector (AMS Biotechnology (UK) Ltd.). This transfervector is a simple derivative (Possee R. D., personal communication) ofthe transfer vector pAcUW2b previously used successfully to preparerecombinant viruses capable of delivering an accelerated biologicaleffect because of the expression of insect selective toxin genes underthe control of the powerful late p10 promoter (Stewart et al. (1991)Nature 352 85-88; McCutchen et al. (1991) Biotechnology 9 848-852).

To release a suitable sKK-0 insert for introduction into pAcUW21 a 20 μgaliquot of the pVL1392/sKK-0 #2.2 plasmid DNA was subject to digestionwith EcoRI and BglII restriction enzymes. In parallel, a 10 μg aliquotof pAcUW21 transfer vector DNA was similarly treated. The restrictiondigests were then run on a preparative 1% agarose/Tris-acetateelectrophoresis gel containing 0.5 μg/ml ethidium bromide. The releasedsKK-0 insert (approx. 282 base pairs) and the linearised vector DNA wereexcised from the gel under UV illumination. They were then recovered bycentrifugation through siliconised glass wool and ethanol precipitation.

Aliquots of the isolated sKK-0 insert and pAcUW21 vector DNA fragmentswere then mixed, together with T4 ligase, in the appropriate bufferconditions (Sambrook et al. (1989) in "Molecular Cloning: A LaboratoryManual" (Cold Spring Harbor Press)). The ligation mixtures were thenused to transform competent DH5α cells by standard methods (Sambrook etal. ibid.). Progeny transformants were selected by overnight growth on Lagar plates containing 100 μg/ml ampicillin. Plasmid DNA was preparedfrom six putative recombinant colonies. Restriction analysis of theseDNAs suggested that all six were pAcUW21 derivatives containing insertswith the size and features expected for sKK-0.

The sequence of the inserts present in two of the above six recombinants(#A1 & #A2) was then checked using a Sequenase™ (USB, Cleveland, Ohio)kit and two synthetic oligonucleotide primers designed to hybridise tothe sKK-0 insert. The sequence of the inserts of both clones matchedprecisely that expected for sKK-0 cloned in the intended manner.

Recombinant #A1, a derivative of transfer vector pAcUW21 containing genesKK-0 under the transcriptional control of the AcMNPV p10 promoter, wastherefore selected for subsequent studies. This recombinant transfervector also possesses an intact AcMNPV polyhedrin gene under the controlof the natural polyhedrin promoter.

EXAMPLE 8

Generation of a Recombinant (Polyhedrin Plus) AcMNPV Carrying a p10Promoter/sKK-0 Gene Expression Unit

Standard baculovirus techniques (King & Possee (1992) "The BaculovirusExpression System: A Laboratory Guide" (Chapman & Hall)) were used togenerate polyhedrin⁺ (occluded) AcMNPV derivatives carrying the sKK-0gene under the transcriptional control of the p10 promoter. This wasaccomplished by co-transfection of Sf21 cells with 200 ng.SauI(Boehringer Mannheim) linearised AcMNPV.lacZ (Possee & Howard (1987)Nucleic Acids Research 15 10233-10248) and 1 μg pAcUW21/sKK-0recombinant #A1 DNA. Candidate recombinant AcMNPV/sKK-0 viruses werethen selected by plaque purification on Sf21 cell monolayers (King &Possee (1992) ibid.) screening initially for inability of viruses tometabolise X-gal (GIBCO/BRL, Grand Island, N.Y.) and the ability toproduce polyhedra (viral occlusion bodies) as judged by microscopy. Sixsuch lacZ- virus (AcMNPV/pAcUW21/sKK-0 #1, AcMNPV/pAcUW21/sKK-0 #2,AcMNPV/pAcUW21/sKK-0 #3, AcMNPV/pAcUW21/sKK-0 #44, AcMNPV/pAcUW21/sKK-0#45 and AcMNPV/pAcUW21/sKK-0 #6) were picked. A second plaque assay wasthen performed with the above six clones on Sf21 cell monolayers topurify them to homogeneity and confirm their inability to synthesiseβgalactosidase and their ability to produce polyhedra.

A small scale cultures of each purified virus was then prepared asdescribed in Example 5. Aliquots of each of these virus stocks were thentaken for bioassay (see Example 9). Parallel aliquots were used toprepare small samples of viral DNA for physical analysis.

Southern blot analyses were performed on the prospectiveAcMNPV/pAcUW121/sKK-0 viral DNAs. These DNAs were treated with: (a)HindIII and (b) EcoRI and BamHI, fractionated by electrophoresis on a 1%agarose gel, transferred to Hybond-N filters and hybridised with anisolated, random-primed α³² P-dCTP labelled, sKK-0 probe.

These studies confirmed that viruses AcMNPV/pAcUW21/sKK-0 #1,AcMNPV/pAcUW121/sKK-0 #4 and AcMNPV/pAcUW21/sKK-0 #5 each contained ansKK-0 gene, with all the anticipated physical features, lacked aβgalactosidase gene and produced polyhedrin⁺ virus particles. VirusAcMNPV/pAcUW21/sKK-0 #1 was selected for all subsequent studies.

EXAMPLE 9

Bioassay of Prospective AcMNPV/pAcUW21/sKK-0 Recombinant Virus

The biological properties of putative AcMNPV/pAcUW21/sKK-0 viruses wereevaluated in a series of injection and oral dosing studies againstH.virescens. Injection assays using purified non-occluded virus stockswere carried out using the method outlined in Example 6. Oral dosingtests with purified polyhedra were undertaken using a modified versionof the droplet feeding method for neonate larvae developed by Hughes andWood ((1981) J.Invertebr. Pathol. 37, 54). Preliminary injection studieson the six putative AcMNPV/pAcUW21/sKK-0 viruses generated as describedin Example 8 indicated that only three clones (AcMNPV/pAcUW21/sKK-0#1,#4 and #5) were carrying the sKK-0 gene. Some of the larvae treatedwith these viruses exhibited abnormal symptoms which initially appearedto be analagous to those observed in insects treated with theAcMNPV/pVL1392/sKK-0 CONO C virus. Subsequent studies onAcMNPV/pCUW21/sKK-0 #1, however, confirmed that these "abnormal" effectswere generally poorly defined and not particularly debilitating and thatAcMNPV/pAcUW21/sKK-0 #1 showed no noticeable advantage over the wildtype AcMNPV in terms of speed of action or insecticidal effect. Oraldosing studies comparing the biological efficacy of AcMNPV/pAcUW21/sKK-0#1 and the wild type AcMNPV confirmed these observations.

EXAMPLE 10

Preparation of AcUW1-PH DNA for the Generation of Recombinant AcMNPV

In light of the different behaviour of polyhedrin minus AcMNPVderivatives in which the sKK-0 gene is expressed from the polyhedrinpromoter (Examples 4,5 & 6) as compared to polyhedrin plus AcMNPVderivatives in which the sKK-0 gene was expressed from the p10 promoter(Examples 7, 8 & 9), we decided to construct a polyhedrin plusderivative in which the sKK-0 gene was expressed from the polyhedrinpromoter to establish if improved insecticidal efficacy could beachieved with the polyhedrin promoter/sKK-0 expression unit but not withthe p10 promoter/sKK-0 expression unit.

The virus selected as a recipient for the polyhedrin promoter/sKK-0expression unit was AcUW1-PH (see Weyer et al. (1990) J.Gen.Virol. 711525-1534 and FIG. 6). This virus carries a p10 promoter/polyhedrin geneexpression unit at the locus occupied by the p10 gene in wild typeAcMNPV and a polyhedrin promoter/lacZ indicator gene expression unit atthe location normally occupied by the polyhedrin gene. It will thereforeform plaques on an Sf21 cell monolayer which contain cells carryingAcMNPV occlusion bodies and which stain blue in the presence of X-Galindicator (GIBCO/BRL, Grand Island, N.Y.). Replacement of the polyhedrinpromoter/lacZ expression unit with the polyhedrin promoter/sKK-0expression unit of the pVL1392/sKK-0 #2.2 recombinant transfer vectorwas therefore anticipated to be a convenient means of preparing apolyhedrin plus AcMNPV derivative carrying a polyhedrin promoter/sKK-0gene expression unit.

A stock of AcUW1-PH virus of unknown titre was kindly provided by Dr R.D. Possee (NERC Institute of Virology and Environmental Microbiology,Mansfield Road, Oxford). Small scale amplification cultures of thisvirus were then prepared by seeding 3 lots of 1.5×10⁶ Sf21 cells in 4ml. TC100/10% foetal calf serum medium (both from GIBCO/BRL) in 25 cm²tissue culture flasks (Bibby, Stone, Staffs.), incubation overnight at28° C., followed by addition of 50 μl, 5 μl and 5 μl of a 1/10 dilutionaliquots of the stock virus to one flask each and continuing incubationat 28 ° C. for a further 6 days. These small scale amplifications weretitered using the standard plaque assay technique (King & Possee (1992)"The Baculovirus Expression System: A Laboratory Guide" (Chapman &Hall)). All plaques obtained contained inclusion bodies and stained bluein the presence of X-Gal. The virus stock produced from the initial 50μlinput culture had a titre of 2.3×10⁷ pfu/ml and was used to producelarger, 200 ml. spinner stock culturess of AcUW1-PH, from which purifiedstocks of virus and subsequently viral DNA were prepared by standardmethods (King & Possee (1992) ibid. & Possee & Howard (1987) NucleicAcids Research 15 10233-10248). A stock of linearised AcUW1-PH DNA wasthen prepared by digestion of 3μg viral DNA with SauI (BoehringerMannheim) according to the manufacturer's recommendations since thisenzyme cleaves only within the βgalactosidase gene and, as withAcMNPV.lacZ, this was anticipated to be a procedure which wouldfacilitate production and detection of recombinant AcMNPV progeny (King& Possee (1992) ibid.).

EXAMPLE 11

Generation of Recombinant (Polyhedrin Plus) AcMNPV Carrying a PolyhedrinPromoter/sKK-0 Gene Expression Unit

Using standard baculovirus techniques (King & Possee (1992) ibid.),recombinant AcMNPV viruses carrying sKK-0 under the transcriptionalcontrol of the polyhedrin promoter, with an active polyhedrin gene, werethen generated by using the SauI linearised AcUW1-PH virus DNA as arecipient for the polyhedrin promoter/sKK-0 expression module frompVL1392/sKK-0 #2.2. This was accomplished by co-transfection of Sf21cells with 200 ng. SauI linearised AcUW1-PH DNA prepared as described inExample 10 and 1 μg pVL1392/sKK-0 #2.2 DNA. Using the plaque assaytechnique, where the viruses generate plaques in Sf21 cell monolayerculture, recombinant virus were selected by initially screening forviruses which lacked the ability to metabolise X-gal (GIBCO/BRL, GrandIsland, N.Y.) but retained the ability to produce occlusion bodies(polyhedra). Six such lacZ- virus (AcUW1-PH/KK0 #1, AcUW1-PH/KK0 #2,AcUW1-PH/KK0 #3, AcUW1-PH/KK0 #4, AcUW1-PH/KK0 #5, AcUW1-PH/KK0 #6) werepicked. A second plaque assay was then performed with each of the abovesix clones on Sf21 to purify them to homogeneity and confirm theirinability to synthesise βgalactosidase and their ability to producepolyhedra. Small scale cultures of each purified virus were thenprepared by the methods outlined in Example 5. Aliquots of each of thesevirus stocks were then taken for bioassay (see Example 12). In parallelaliquots of the small scale virus stocks were used to prepare smallsamples of viral DNA for physical analysis.

Southern blot analysis were performed on the prospectiveAcUW1-PH/pVL1392/sKK-0 viral DNAs. These DNAs were treated with BglIIand BscI (an isoschizomer of ClaI), fractionated by electrophoresis on a1.4% agarose gel, transferred to Hybond-N filters and hybridised with anisolated, random-primed α³² P-dCTP labelled, sKK-0 probe. These studiesconfirmed that viruses AcUW1-PH/sKK-0 #2, AcUW1-PH/sKK-0 #4,AcUW1-PH/sKK-0 #5 and AcUW1-PH/sKK-0 #6 each contained an sKK-0 gene,with all the anticipated physical features, lacked a βgalactosidase geneand produced polyhedrin virus particles.

Virus AcUW1-PH/sKKO #2 was selected for subsequent studies.

EXAMPLE 12

Bioassay of Prospective AcUW1-PH/pVL1392/sKK-0 Recombinant Virus

The biological efficacy of putative AcUW1-PH/pVL1392/sKK-0 recombinantviruses was evaluated in a series of injection and oral dosing studiesagainst Heliothis virescens larvae. Injection assays with purifiednon-occluded virus stocks were undertaken using the methods described inExample 6. Oral dosing studies with purified polyhedra were carried outusing a modified version of the droplet feeding assay described inExample 9.

Preliminary injection studies comparing the biological activity of thesix putative AcUW1-PH/pVL1392/sKK-0 viruses generated as described inExample 11 indicated that only viruses AcUW1-PH/sKK-0 #2, AcUW1-PH/sKK-0#4, AcUW1-PH/sKK-0 #5 and AcUW1-PH/sKK-0 #6 might have an enhancedinsecticidal effect compared to the wild type virus.AcUW1-PH/pVL1392/sKK-0 #2 was selected for scale-up and furthercharacterisation.

Injection assay data indicated that larvae treated with theAcUW1-PH/pVL1392/sKK-0 #2 virus demonstrated abnormal symptomology from72 HAT onwards (Table IV). By 96 HAT the majority of larvae were deadand the only survivor was paralysed. All larvae were dead by 144 HAT. Incontrast, no abnormal effects were observed in larvae treated with wildtype AcMNPV until 120 HAT when 50% of larvae were dead; 100% mortalitywas recorded for the wild type AcMNPV at 144 HAT.

A series of oral dosing studies was carried out to compare thebiological efficacy of AcUW1-PH/pVL1392/sKK-0 and the wild type AcMNPVusing purified polyhedra. In all cases, the AcUW1-PH/pVL1392/sKK-0 #2was shown to have a significantly faster speed of kill than the wildtype and hence an improved insecticidal effect. Larvae treated with theAcUW1-PH/pVL1392/sKK-0 virus started to show symptoms of paralysis anddeath from 72 HAT onwards (Table V). Probit analysis of the doseresponse data confirmed that the speed of kill of AcUW1-PH/pVL1392/sKK-0#2 was significantly faster than that of the wild type AcMNPV at 72 and96 HAT but that by 120 HAT the wild type virus had caught up.

The baculovirus of the present invention may be applied to the insect orlocus of the insect in accordance with the known, or developed,techniques of the art. Preferably, the baculovirus is formulated. Any ofthe known, or developed, formulation may be used as appropriate. Theformulations should be optimised to maximise speed of kill, andpreferably protect the baculovirus from u.v. radiation.

Baculoviruses may be applied by air (particularly against forest pests),by a boom type sprayer for agricultural crops, or by high pressureequipment primarily for fruit and vegetable crops. They are generallyapplied by spray rather than in dust or granule formulation, thewettable powders being a preferred means of application. Adjuvants, forexample surface active agents such as spreaders, stickers andemulsifiers, sunlight screens, buffers and also gustatory stimulants,may be added.

The fact that homologous recombination has occurred in accordance withthe present invention can be ascertained using conventional methods. Inparticular, the precise change in the viral genome can be investigatedusing, for example endonuclease restriction or sequencing. Any changesin host range can also be assessed using known bioassay methods. It isalso possible to follow the genetic make-up of viruses using a genomicmarker.

Initial investigations into genetic exchange between engineered virusesand other viruses, or even acquisition of host cell DNA should be heldin the laboratory, in which a group of larvae is infected with bothwild-type and recombinant virus. Additional larvae can be added asrepresentatives of successive generations.

Further investigations can be made using contained field trials. In suchtrails, larvae are infected in the laboratory before being introducedinto the field, where the virus population is monitored over time.

                  TABLE I    ______________________________________    Preliminary assessment of the biological activity of synthetic    preparations of KK-0, KK-1 AND KK-2 proteins by injection into    Heliothis virescens larvae.sup.1           Dose    Treatment           per insect                    Effects    ______________________________________    KK-0   1 μl  Loss of coordination within 30 secs post-                    injection. All larvae showing flaccid paralysis                    within 2-4 mins of injection & unable to stand,                    reinvert or respond to stimulation; limp & pliable.                    Effects began to wear off after 4-6 minutes &                    most larvae appeared normal at 10 mins post-                    injection. All larvae alive & well at 24 HAT.           2 μl  Symptoms as above but more severe & pro-                    longed. Gradual recovery with all larvae appear-                    ing normal at 45 mins after injection. All alive &                    well at 24 HAT.    KK-1   1 μl  Symptoms first observed within 30 secs of                    injection. Most larvae showing flaccid paralysis                    within 2-4 minutes: symptoms as for KK-0.                    Effects were short-lived & all larvae appeared                    normal at 6-10 mins post-injection. All larave                    alive & normal at 24 HAT.    KK-2   1 μl  No adverse effects observed even at 24 HAT.    Control.sup.2           --       No adverse effects observed during experiment.    ______________________________________     .sup.1 Six fifthinstar larvae injected per treatment; average body mass:     300 mg     .sup.2 Control: 2 μl 0.1M ammonium bicarbonate

                  TABLE II    ______________________________________    Preliminary assessment of the biological activity of synthetic    preparations    of KK-0, KK-1 and KK-2 proteins against Periplaneta americana.sup.1    by injection.           Dose    Treatment           per insect                    Effects    ______________________________________    KK-0   1 μl  Temporary paralysis of back legs observed within                    1 min post-injection but effects had disappeared                    by 6 mins pi. Capable of responding normally to                    stimulation but locomotion slow & poorly coordi-                    nated from 20 min-3 h pi. Moribund by 21 HAT                    & dead at 22 HAT.           5 μl  Tremoring followed by paralysis of back legs                    within 30 secs pi. Knocked down with mid- &                    hind legs paralysed by 20 min pi but front legs                    still working normally. Complete paralysis within                    1 HAT. No recovery - moribund at 21 HAT &                    dead at 22 HAT.           10 μl Tremoring & loss of coordination initially in back                    legs but rapidly spreading to mid- & front legs                    followed by total paralysis. No recovery -                    moribund/dead at 22 HAT.    KK-1   1 μl  Tremors followed by loss of movement in hind                    legs within 15 sec pi; front legs still actively                    moving at 5 min pi. Moribund at 20 min pi. Other                    symptoms included back arching. Some recovery                    between 1.5-3 HAT. At 21 HAT, insects knocked                    down & unable to reinvert. No recovery by 52                    HAT.           5 μl  Loss of coordination & paralysis in hind legs                    immediately after injection, rapidly spreading to                    mid- & front legs. Complete paralysis within 5                    min pi. No recovery & insects dead at 21 HAT.           10 μl Rapid loss of coordination followed by complete                    paralysis within 30 secs pi. Occasional tremors                    observed thereafter but insects remained knocked                    down for remainder of experiment. No recovery                    & all dead at 21 HAT.    KK-2   1 μl  No abnormal effects observed throughout                    experiment.           2 μl  Abnormal symptomology included arching back                    & poorly coordinated locomotion within 1 min pi.                    Some recovery observed from 20 min pi onwards                    but insects dead at 69 HAT.           5 μl  Temporary loss of coordination immediately after                    injection; other symptoms included dorsal                    arching. Insects appeared to have recovered by                    1.5 HAT but were dead at 45 HAT.    Control.sup.2           --       No abnormal effects observed throughout                    experiment.    ______________________________________     .sup.1 Adult male roaches with an average body mass of 840 mg     .sup.2 Control: 10 μl 0.1M ammonium bicarbonate solution

                                      TABLE III    __________________________________________________________________________    Evaluation of the biological efficacy of AcMNPV/pVL1392/sKK-0 CONO C by    injection    into Heliothis virescens larvae.sup.6              Number dead (+ affected)    Treatment.sup.1              24 hr                 48 hr                    72 hr  96 hr    120 hr                                          144 HR    __________________________________________________________________________    AcMNPV/pVL1392/              0/10                 0/10                    0/10   0/10     1/10   6/10    lacZ.sup.1    AcMNPV/pVL1392/              0/11                 0/11                    0/11   3/11     5/11  11/11    sKK-0 Cono C.sup.1                    (+3FP.sup.3, 1PP.sup.4)                           (+1FP, 3PP, 4AF.sup.5)                                    (+5FP, 1PP)    Control.sup.2              0/7                 0/7                    0/7    0/7      0/7   1/7    __________________________________________________________________________     .sup.1 Dose rate = 1.5 × 10.sup.4 pfu/larva.     .sup.2 Control = Sterile water.     .sup.3 FP  Full flaccid paralysis: insect is moribund, cannot stand, walk     or reinvert & is capable only of limited feeble movements of mouthparts     and claspers in response to stimulation; limp & pliable.     .sup.4 PP  Partial paralysis: mid/posterior part of body is partially     paralysed; larva is still capable of some locomotory activity and can     reinvert but movements are poorly coordinated.     .sup.5 AF  Affected: larva responds slowly to stimulation but is     unable/unwilling to walk or feed.     .sup.6 Fourthinstar larvae; average body mass = 100 mg.

                  TABLE IV    ______________________________________    Evaluation of the biological efficacy of AcUW1-PH/pVL1392/sKK-0 #2    and AcMNPV wild type viruses by injection into Heliothis virescens    larvae.sup.6.            Number of insects dead (+ affected)    Treatment.sup.1              1      2      3        4     5   6   7    ______________________________________    AcMNPV wt 0/6    0/6    0/6      0/6   3/6 6/6 6/6    AcUW1-PH/ 0/6    0/6    0/6      5/6   6/6 6/6 6/6    pVL1392/KK0-2           (+1PP.sup.4, 1AF.sup.5)                                     (+1FP.sup.3)    AcUW1-PH/ 0/7    0/7    0/7      0/7   0/7 3/7 7/7    pVL1392/lacZ    Control.sup.2              0/5    0/5    0/5      0/5   0/5 0/5 0/5    ______________________________________     .sup.1 Dose = .sup.˜ 1 × 10.sup.4 pfu/larva     .sup.2 Control: Sterile water     .sup.3 FP  flaccid paralysis; moribund and cannot stand, reinvert, walk o     respond normally to stimulation; only sign of life indicated by very     feeble movements of mouthparts/claspers in response to stimulation.     .sup.4 PP  partial paralysis; cannot reinvert or walk; mid & posterior     part of body is completely immobilised but anterior portion of body can     still move & pro and falselegs can still be withdrawn in response to     stimulation.     .sup.5 AF  affected; insect can stand, reinvert & respond normally to     stimulation but is incapable of coordinated locomotion.     .sup.6 Fifthinstar larvae, average body mass = 300 mg.

                                      TABLE V    __________________________________________________________________________    Evaluation of the biological efficacy of AcUW1-PH/pVL1392/sKK-0 #2 and    AcMNPV    wild type viruses against H. virescens larvae by oral dosing.sup.1            Dose  % kill (+ % affected.sup.2)    Treatment            (PIBs/ml)                  72 hr                      96 hr                          120 hr                               144 hr                                    168 hr    __________________________________________________________________________    AcMNPV WT            1.0 × 10.sup.7                  3   33  67   70   70            3.3 × 10.sup.6                  0    7  17   20   20            1.1 × 10.sup.6                  0   13  20   20   20            3.7 × 10.sup.5                  0    0   7   10   10            1.2 × 10.sup.5                  0    0   3    3    3            4.1 × 10.sup.4                  0    0   4    4    4    AcUW1-PH            1.0 × 10.sup.7                  7   37  67   73   73    pVL1392           (+30)    sKK-0 #2            3.3 × 10.sup.6                  3   17  31   31   34                      (+10)            1.1 × 10.sup.6                  4   18  32   32   32                  (4)  (+7)            3.7 × 10.sup.5                  3   10  14   14   14                       (+3)            1.2 × 10.sup.5                  7   10  13   17   20                  (+3)    (+3) (3)            4.1 × 10.sup.4                  3    3   3    3    3    Control --    0    0   0    0    0    __________________________________________________________________________    Time after          AcMNPV wild type                         AcUW1-PH/pVL1392/sKK-0 #2    treatment          LC.sub.50.sup.3                95% C.I. LC.sub.50                               95% C.I.    __________________________________________________________________________     96   --    --       6.1 × 10.sup.6                               3.3 × 10.sup.6 -1.7 × 10.sup.7    120   7.6 × 10.sup.6                4.4 × 10.sup.6 -1.9 × 10.sup.7                         5.2 × 10.sup.6                               2.7 × 10.sup.6 -1.6 × 10.sup.7    144   6.6 × 10.sup.6                3.9 × 10.sup.6 -1.5 × 10.sup.7                         4.3 × 10.sup.6                               2.3 × 10.sup.6 -1.2 × 10.sup.7    168   6.6 × 10.sup.6                3.9 × 10.sup.6 -1.5 × 10.sup.6                         4.0 × 10.sup.6                               2.2 × 10.sup.6 -1.0 × 10.sup.7    __________________________________________________________________________     .sup.1 Neonate larvae dosed using a modified version of the droplet     feeding assay (Hughes & Wood, 1981); 30 larvae/dose.     .sup.2 () % paralysed larvae     .sup.3 LC.sub.50 values (PIBs/ml virus suspension) estimated using a     standard logit dose response procedure (Ashton, 1972).

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 18    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 269 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 17..250    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TTAGATCTAATTCACCATGAAGCTGACATGTATGATGATCGTGGCCGTG49    MetLysLeuThrCysMetMetIleValAlaVal    1510    CTGTTCCTGACCGCCTGGACCTTCGCCACTGCAGACGATCCCCGCAAC97    LeuPheLeuThrAlaTrpThrPheAlaThrAlaAspAspProArgAsn    152025    GGCCTGGGCAACCTGTTCTCCAACGCCCACCACGAAATGAAGAACCCC145    GlyLeuGlyAsnLeuPheSerAsnAlaHisHisGluMetLysAsnPro    303540    GAGGCATCCAAGCTTAACAAGCGCTGGTGTAAGCAGTCCGGAGAGATG193    GluAlaSerLysLeuAsnLysArgTrpCysLysGlnSerGlyGluMet    455055    TGTAACCTGCTGGACCAGAACTGTTGTGACGGCTACTGTATCGTGCTG241    CysAsnLeuLeuAspGlnAsnCysCysAspGlyTyrCysIleValLeu    60657075    GTGTGCACCTAGTGACGGCCGGATCCTT269    ValCysThr    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 78 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla    151015    TrpThrPheAlaThrAlaAspAspProArgAsnGlyLeuGlyAsnLeu    202530    PheSerAsnAlaHisHisGluMetLysAsnProGluAlaSerLysLeu    354045    AsnLysArgTrpCysLysGlnSerGlyGluMetCysAsnLeuLeuAsp    505560    GlnAsnCysCysAspGlyTyrCysIleValLeuValCysThr    657075    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 84 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TTAGATCTAATTCACCATGAAGCTGACATGTATGATGATCGTGGCCGTGCTGTTCCTGAC60    CGCCTGGACCTTCGCCACTGCAGA84    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 89 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GGATGCCTCGGGGTTCTTCATTTCGTGGTGGGCGTTGGAGAACAGGTTGCCCAGGCCGTT60    GCGGGGATCGTCTGCAGTGGCGAAGGTCC89    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 89 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CACGAAATGAAGAACCCCGAGGCATCCAAGCTTAACAAGCGCTGGTGTAAGCAGTCCGGA60    GAGATGTGTAACCTGCTGGACCAGAACTG89    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 73 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    AAGGATCCGGCCGTCACTAGGTGCACACCAGCACGATACAGTAGCTACAGTAGCCGTCTG60    GTCACTAGGTTAC73    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    TTAGATCTAATTCACCATGAAGCTGACATG30    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    AAGGATCCGGCCGTCACTAGGTGCAC26    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GATCAGATCTGCAGCGGCCGCTCCAGAATTCTAGAAGGTACCCGG45    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GATCCCGGGTACCTTCTAGAATTCCGGAGCGGCCGCTGCAGATCT45    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    TATAAATATGCCGGATTAT19    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 49 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    TATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCC49    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CTGTTTTCGTAACAG15    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    CGGATTTCCTTGAAG15    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 282 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGCGCGGATCAGATCTAATTCACCATGAAGCTGACATGTATGATGATCGTGGCCGTGCTG60    TTCCTGACCGCCTGGACCTTCGCCACTGCAGACGATCCCCGCAACGGCCTGGGCAACCTG120    TTCTCCAACGCCCACCACGAAATGAAGAACCCCGAGGCATCCAAGCTTAACAAGCGCTGG180    TGTAAGCAGTCCGGAGAGATGTGTAACCTGCTGGACCAGAACTGTTGTGACGGCTACTGT240    ATCGTGCTGGTGTGCACCTAGTGACGGCCGCTCCAGAATTCT282    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 78 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla    151015    TrpThrPheAlaThrAlaAspAspProArgAsnGlyLeuGlyAsnLeu    202530    PheSerAsnAlaHisHisGluMetLysAsnProGluAlaSerLysLeu    354045    AsnLysArgTrpCysLysGlnSerGlyGluMetCysAsnLeuLeuAsp    505560    GlnAsnCysCysAspGlyTyrCysIleValLeuValCysThr    657075    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 77 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla    151015    TrpThrPheAlaThrAlaAspAspSerSerAsnGlyLeuGluAsnLeu    202530    PheSerLysAlaHisHisGluMetLysAsnProGluAlaSerLysLeu    354045    AsnLysArgCysIleGluGlnPheAspProCysGluMetIleArgHis    505560    ThrCysCysValGlyValCysPheLeuMetAlaCysIle    657075    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 77 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    MetLysLeuThrCysMetMetIleValAlaValLeuPheLeuThrAla    151015    TrpThrPheValThrAlaAspAspSerGlyAsnGlyLeuGluAsnLeu    202530    PheSerLysAlaHisHisGluMetLysAsnProGluAlaSerAsnLeu    354045    AsnLysArgCysAlaProPheLeuHisProCysThrPhePhePhePro    505560    AsnCysCysAsnSerTyrCysValGlnPheIleCysLeu    657075    __________________________________________________________________________

I claim:
 1. A recombinant baculovirus having a genome which comprises apolyhedrin gene and a heterologous gene, which expresses an insecticidalprotein, wherein the polyhedrin gene and the heterologous gene are notunder the control of the same promoter and at least 10% of the genome ofthe baculovirus separates the polyhedrin gene and the heterologous genesuch that viral progeny produced by a recombination event with wild-typebaculovirus which are viable progeny produced by a recombination eventwith wild-type baculovirus which are viable do not retain expression ofboth the polyhedrin gene and the heterologous gene.
 2. A recombinantbaculovirus according to claim 1 wherein at least one region of thegenome which is homologous to a region of the genome of the wild-typevirus occurs between the polyhedrin gene and the heterologous gene.
 3. Arecombinant baculovirus according to claim 1 wherein the polyhedrin geneand heterologous gene are located between two regions of the genomewhich regions are homologous to regions of the genome of the wild-typevirus, wherein the location lacks an essential gene which is presentbetween the homologous regions in the wild-type virus.
 4. A recombinantbaculovirus according to claim 1 wherein the polyhedrin gene is underthe control of the p10 promoter.
 5. A recombinant baculovirus accordingto claim 1 wherein the heterologous gene is under the control of thepolyhedrin promoter.
 6. A recombinant baculovirus according to claim 1wherein the genome has been modified to disable or delete the p10 gene.7. A recombinant baculovirus according to claim 1 wherein the separationis about 12% of the genome.
 8. A recombinant baculovirus according toclaim 1 wherein the polyhedrin gene and the heterologous gene areseparated by at least 13 kilo base pairs.
 9. A recombinant baculovirusaccording to claim 8 wherein the separation is about 15 kilo base pairs.10. A recombinant baculovirus according to claim 1 wherein thepolyhedrin gene and the heterologous gene are located in the samerelationship to each other as they are in the construct shown in FIG.6b.
 11. The recombinant baculovirus according to claim 10 which is shownin FIG. 6b.
 12. A progeny baculovirus of a baculovirus recombinantaccording to claim
 1. 13. A method of combating insect pests at a locuswhich comprises treating the pests or locus with a recombinantbaculovirus according to claim 1.